Target Affinity and Structural Analysis for a Selection of Norovirus Aptamers

Figures

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  Figure 1. VLP-binding graphs of aptamers M1 (A), M6-2 (B), SMV19 (C), SMV21 (D), Buf-2 (E), AG3 (F) and Beier (G). The different curves represent the NoV VLP genotypes GI.1 (yellow), GI.7 (black), GII.3 (red), GII.4 New Orleans (blue), and GII.4 Sydney (green). All graphs are shown with the y-axes indicating bound DNA to target protein from 0 to 80% to allow uniform evaluation of the graphs.

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  Figure 2. CD spectra of aptamer candidates M1, M6-2, SMV19, SMV21, Buf-2, AG3, and Beier (black curve) and the same biotinylated candidates (blue curve) are shown in (A–G), respectively.

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  Figure 3. Aptamer mediated dot-blot using selected aptamers and five different genotypes of VLPs. Dot-blot with M1, M6-2, SMV19, SMV21, Buf-2, AG3, and Beier are depicted in (A–G), respectively. The dot-blot negative control (NC), omitting an aptamer binding step, showed no spots on the membrane (H). The concentration of the different VLP solutions applied to the nitrocellulose membrane (0–1500 nM) is indicated on each row’s left side. The different NoV genotypes used are indicated by the abbreviations GI.1, GI.7, GII.3, and GII.4 Sydney (Syd.) and New Orleans (Nola).

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  Figure 4. Aptamer-mediated NoV pull-down from stool suspension using different aptamers. The Ct values are shown on the y-axis, and each bar on the x-axis represents the aptamer used during the pull-down. Statistically significant differences compared to the negative control (NC) are indicated with asterisks above each bar. The p-value indication for the asterisks is: not significant (ns) for p-value > 0.05, two asterisk (**) for p-values between of 0.0021–0.0332 and four asterisks (****) for p-values < 0.0001. The aptamer mediated pull-downs were completed for each aptamer in triplicate from a 100-fold dilution from partially purified stool (A) and 1000-fold dilution of the same preparation (B). In (B) no Ct values were obtained for NC and AG3. Therefore, the maximum number of cycles run in the PCR program (45) is shown.